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Purity: ≥98%
DAPT (also known as GSI-IX; LY-374973) is a novel, potent and selective γ-secretase inhibitor used in the study of the Notch signaling pathway and is reported to be able to reduce the levels of beta-amyloid in a mouse model of Alzheimer's disease. Through its indirect inhibition of Notch, a substrate of γ-secretase, DAPT inhibits the production of Aβ in HEK 293 cells, with an IC50 of 20 nM.
| Targets |
Aβ (IC50 = 115 nM); Aβ42 (IC50 = 200 nM)
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|---|---|
| ln Vitro |
DAPT also inhibits the production of Aβ in human primary neuronal cultures, with an IC50 for Aβ total and Aβ42 of 115 nM and 200 nM, respectively. These values are 5–10 times lower than those found in HEK 293 cells.[1] According to a recent study, DAPT has an IC50 of 11.3 μM and inhibits SK-MES-1 cell proliferation in a concentration-dependent manner. Furthermore, by blocking the Notch receptor signaling pathway, DAPT causes both caspase-dependent and caspase-independent apoptosis in lung squamous cell carcinoma cells.[2]
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| ln Vivo |
DAPT administration (100 mg/kg) results in a strong and long-lasting pharmacodynamic effect in PDAPP mice. Within an hour of administration, brain DAPT levels surpass 100 ng/g, and they continue to rise for up to 18 hours, peaking at 490 ng/g after 3 hours. Additionally, at that time, DAPT (100 mg/kg) also causes a 50% reduction in cortical total Aβ and Aβ42 in a dose-dependent manner.[1] In the cerebral cortexes of rats, DAPT (40 mg/kg) increases cell apoptosis in response to prolonged neuroinflammation and suppresses the γ-secretase activity induced by LPS.[3]
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| Enzyme Assay |
For standard Aβ reduction assays, human embryonic kidney cells (American Type Culture Collection CRL-1573) transfected with the APP751 gene (HEK 293) are utilized. In Dulbecco's modified Eagle medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, cells are plated in 96-well plates and left to adhere for the duration of the night. To achieve a final concentration of 0.1% DMSO in media, DAPT are diluted from stock solutions in dimethylsulfoxide (DMSO). Fresh compound solutions are applied after the cells have been pre-treated with DAPT for two hours at 37 °C. The media is then aspirated off. Following a further two-hour treatment period, the conditioned medium is removed and subjected to a sandwich ELISA (266–3D6) that is specifically designed to detect total Aβ. Decrease in Aβ production is expressed as a percentage inhibition and quantified in relation to control cells treated with 0.1% DMSO. Potency is calculated using XLfit software by fitting data from at least six doses in duplicate to a four-parameter logistical model. Before being used, more than 90% of the neurons in the neuronal cultures of PDAPP mice and humans were shown to have matured in serum-free medium. After adding new media to each well and incubating for 24 hours at 37 °C without DAPT, conditioned media is collected to establish baseline Aβ values. After treating cultures for a further 24 hours at 37 °C in fresh media containing DAPT at the appropriate range of concentrations, conditioned media is collected. The same ELISA (266–3D6) that is used for the HEK 293 cell assays is used to analyze samples for total Aβ measurement. A distinct ELISA (21F12–3D6) that uses a capture antibody specific for the Aβ42 C-terminus is used to analyze samples for Aβ42 production. The difference between the values for the compound treatment and baseline periods determines inhibition of production for both total Aβ and Aβ42. Potency is ascertained by using the XLfit software to analyze data after charting percentage inhibition against DAPT concentration.
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| Cell Assay |
The cells are plated in 96-well plates and left to react for 72 hours with either 0.1% DMSO or DAPT at concentrations between 2.5 μM and 160 μM. With a few minor adjustments, the 3-(4, 5)-dimethylthiahiazo-(-z-y1)-3, 5-di-phenytetrazoliumromide (MTT) dye reduction assay is used to determine cytotoxicity. To summarise, following DAPT incubation, 180 μL of medium in each well is mixed with 20 μL of MTT solution (5 mg/mL in PBS), and the plates are then incubated for 4 hours at 37 °C. Finally, 150 μL of DMSO is added to each well and shaken at room temperature for 15 minutes. Absorbance values are obtained by measuring absorption using an enzyme-linked immunosorbent assay at 490 nm. α-MEM is used as the blank solution, supplemented with the same volume of MTT solution and solvent. With SPSS, the PROBIT program is used to calculate the IC50 value.
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| Animal Protocol |
Mice: The APPV717F mutant form of the amyloid precursor protein is overexpressed in the three- to four-month-old heterozygous PDAPP transgenic mice. Equal numbers of age-matched male and female animals are fasted overnight before treatment begins in each of the ten treatment groups. Doses of 10 mL/kg are given to the treatment and control groups using either DAPT or vehicle alone. All Aβ and APP measurements are taken after the tissues have been processed. Once the brain is removed, one hemisphere's cortex is homogenized, centrifuged, and the Aβ measurements are performed on the supernatant. In order to analyze compound levels, the cortex from the opposite hemisphere is snap frozen. Aβ levels are measured in nanograms per gram of wet tissue weight, and percentage reductions are computed in relation to the average Aβ level of tissue from control animals that were given no medication. Non-parametric Mann-Whitney statistics are used to analyze data and determine significance.
Rats: Rats (260–290 g) that are male Sprague-Dawleys are employed. With MCAO, the DAPT solution is injected stereotactically into the lateral cerebral ventricle (LV). Coordinates of −0.8 mm anteroposterior, ±1.5 mm mediolateral, and −4.5 mm dorsoventral from the bregma are used for the stereotactic injections into the LVs. thirty rats are divided into three operating groups at random (10 rats in each group): the DAPT group, which receives DAPT as 0.03 mg/kg after MCAO, the sham-operated group, which receives an equal volume of PBS without MCAO operation, and the MCAO group, which receives an equal volume of PBS after MCAO operation. The first neurological function is evaluated 24 hours after the operation, and the second neurological function is evaluated 48 hours later. In the meantime, measurements and comparisons between various groups are made for brain water content and infarction volume.
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| References | |
| Additional Infomation |
DAPT is a dipeptide consisting of alanylphenylglycine derivatised as a 3,5-difluorophenylacetamide at the amino terminal and a tert-butyl ester at the carboxy terminal. It is a gamma-secretase inhibitor. It has a role as an EC 3.4.23.46 (memapsin 2) inhibitor. It is a dipeptide, a difluorobenzene, a carboxylic ester and a tert-butyl ester.
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| Molecular Formula |
C23H26F2N2O4
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|---|---|
| Molecular Weight |
432.46
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| Exact Mass |
432.186
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| Elemental Analysis |
C, 63.88; H, 6.06; F, 8.79; N, 6.48; O, 14.80
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| CAS # |
208255-80-5
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| Related CAS # |
DAPT;208255-80-5
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| PubChem CID |
5311272
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| Appearance |
White to off-white solid powder
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| Density |
1.2±0.1 g/cm3
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| Boiling Point |
612.2±55.0 °C at 760 mmHg
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| Flash Point |
324.1±31.5 °C
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| Vapour Pressure |
0.0±1.8 mmHg at 25°C
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| Index of Refraction |
1.535
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| LogP |
3.98
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
9
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| Heavy Atom Count |
31
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| Complexity |
622
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| Defined Atom Stereocenter Count |
2
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| SMILES |
FC1C([H])=C(C([H])=C(C=1[H])C([H])([H])C(N([H])[C@@]([H])(C([H])([H])[H])C(N([H])[C@]([H])(C(=O)OC(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H])C1C([H])=C([H])C([H])=C([H])C=1[H])=O)=O)F
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| InChi Key |
DWJXYEABWRJFSP-XOBRGWDASA-N
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| InChi Code |
InChI=1S/C23H26F2N2O4/c1-14(26-19(28)12-15-10-17(24)13-18(25)11-15)21(29)27-20(16-8-6-5-7-9-16)22(30)31-23(2,3)4/h5-11,13-14,20H,12H2,1-4H3,(H,26,28)(H,27,29)/t14-,20-/m0/s1
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| Chemical Name |
tert-butyl (2S)-2-[[(2S)-2-[[2-(3,5-difluorophenyl)acetyl]amino]propanoyl]amino]-2-phenylacetate
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| Synonyms |
LY-374973; DAPT; LY 374973; LY374973; GSI-IX; DAPT peptide; GSI-IX; GSI IX; N-(2FPhAc)Ala-phenyl-Gly t-butyl ester; 208255-80-5; DAPT (GSI-IX); GSI-IX; gamma-Secretase Inhibitor IX; C23H26F2N2O4; tert-butyl (2S)-2-[[(2S)-2-[[2-(3,5-difluorophenyl)acetyl]amino]propanoyl]amino]-2-phenylacetate; CHEBI:86193;
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (5.78 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: 2.5 mg/mL (5.78 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (5.78 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. Solubility in Formulation 4: ≥ 1 mg/mL (2.31 mM) (saturation unknown) in 10% EtOH + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 10.0 mg/mL clear EtOH stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of normal saline to adjust the volume to 1 mL. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. Solubility in Formulation 5: 4% DMSO+corn oil: 10 mg/mL Solubility in Formulation 6: 10 mg/mL (23.12 mM) in Corn Oil (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. Solubility in Formulation 7: 5 mg/mL (11.56 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.3124 mL | 11.5618 mL | 23.1235 mL | |
| 5 mM | 0.4625 mL | 2.3124 mL | 4.6247 mL | |
| 10 mM | 0.2312 mL | 1.1562 mL | 2.3124 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
| NCT Number | Recruitment | interventions | Conditions | Sponsor/Collaborators | Start Date | Phases |
| NCT03606642 | Active Recruiting |
Drug: Dual Antiplatelet (DAPT) Therapy Device: The Synergy® stent |
Atherosclerosis Stent Placement |
HonorHealth Research Institute | November 19, 2018 | Phase 2 |
| NCT04135677 | Recruiting | Drug: DAPT Drug: Rivaroxaban |
Thrombi Stroke |
Shanghai Zhongshan Hospital | November 11, 2022 | Phase 4 |
| NCT03462498 | Active Recruiting |
Drug: 1-months DAPT Drug: 12-month DAPT |
Coronary Artery Disease Acute Coronary Syndrome |
Kyoto University, Graduate School of Medicine |
April 2, 2018 | Phase 4 |
| NCT06013020 | Recruiting | Drug: NOAC+DAPT Drug: DAPT |
STEMI - ST Elevation Myocardial Infarction |
Zunyi Medical College | August 28, 2023 | Phase 4 |
| NCT02619760 | Active Recruiting |
Drug: 1-month DAPT Drug: 12-month DAPT |
Coronary Artery Disease | Kyoto University, Graduate School of Medicine |
December 2015 | Phase 4 |
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